The current treatment options for metastatic colon carcinoma are limited. Our preclinical research indicates that immune-modulatory gene therapy may be effective for the treatment of advanced metastatic colorectal cancer. Using an orthotopic murine model for pre-established hepatic colon carcinoma, we have shown that intratumoral injection of an adenoviral vector expressing murine interleukin-12 (Adv/mIL-12) and 4-1BB, a co-stimulatory molecule normally found on activated dendritic and T cells, can induce dendritic cell and CD8+ T cell activation and proliferation. This leads to tumor regression and prolonged survival of treated mice. However, this therapeutic effect is significantly compromised by large tumor burdens (>8x8-10x10mm2). Significant accumulation of myeloid suppressor cells (MSC) and T regulatory cells (Treg) were found in animals bearing large tumors. The lack of proper antigen presentation and Treg mediated immune suppression are two of the main means by which tumor cells evade the immune system and present the major obstacles for cancer immune therapy. We found that CD 40 engagement greatly improved the long-term survival rate of mice bearing large tumors when used in conjunction with IL-12 + anti-4-lBB combination therapy. Furthermore, agonistic anti-CD40 antibody prevented MSC-mediated Treg induction. We hypothesize that CD40 activation by agonistic antibody or Ig-CD40 ligand fusion protein can enhance anti-tumor responses through multiple mechanisms: 1) CD40 ligation can prevent Treg induction through CD40 signaling on MSC;2) CD40 activation can induce the differentiation of MSC into dendritic cells and downregulate the inhibitory receptors, PIR-B (paired Ig-like receptor B) and gp49b, which are highly expressed on MSC and immature DCs. Three specific aims will be pursued in order to understand the mechanisms underlying the CD40 mediated regulation of MSC function and reversion of immune suppression in the large tumor setting: 1) Study the mechanisms underlying the prevention of MSC-induced Treg development by anti-CD40 ligation;2) Investigate the negative receptor signals as the potential mechanism behind the ineffective anti-tumor response to synergistic IL-12 and 4-1BB activation in mice with large tumor burdens;3) Determine the therapeutic effect of blocking negative inhibitory receptors and reversing immune suppression by co-stimulatory molecules on the persistence of an effective anti-tumor response following immune activation in the large tumor bearing host. The completion of these studies will not only provide a new insight into the mechanisms of tumor mediated suppression and tolerance, but also facilitate effective clinical translation of our immune enhancing therapy.